Abel Salazar 2, 4000 Porto, Portugal Received 26 May 1999; accepted 12 August 1999 Abstract - The G-banding technique was performed on chromosomes from gill tissue of three cupped oyster
Trang 1Original article
Alexandra Leitão a Catherine Thiriot-Quiévreux
Pierre Boudr Isabel Malheiro
a
Observatoire océanologique, Université P et M Curie, CNRS-INSU,
BP 28, 06230 Villefranche-sur-Mer, France
b
Laboratoire de génétique et pathologie, Station Ifremer,
BP 133, 17390 La ’Iremblade, France
c
Instituto de ciências biomédicas de Abel Salazar, Universidade do Porto,
Lg Prof Abel Salazar 2, 4000 Porto, Portugal
(Received 26 May 1999; accepted 12 August 1999)
Abstract - The G-banding technique was performed on chromosomes from gill
tissue of three cupped oyster species: Crassostrea gigas, Crassostrea angulata and Crassostrea virginica Identification of the ten individual chromosome pairs was
obtained Comparative analysis of G-banded karyotypes of the three species showed
that their banding patterns generally resembled each other, with chromosome pair 3
being similar in all three species However, differences from one species to another were
also observed The G-banding pattern highlighted greater similarities between C gigas
and C angulata than between these two species and C virginica, thus providing an
additional argument for genetic divergence between these two evolutionary lineages.
C gigas and C angulata showed a different G-banding patterns on the two arms of chromosome pair 7, which agrees with their taxonomic separation The application of this banding technique offers a new approach to specific problems in oyster taxonomy and genetics © Inra/Elsevier, Paris
chromosome / G-banding / Crassostrea gigas / Crassostrea angulata / Crassostrea
virginica
*
Correspondence and reprints
E-mail: thiriot@ccrv.obs-vlfr.fr
Trang 2Résumé - Étude du marquage chromosomique bandes G chez trois espèces
d’huîtres creuses : Crassostrea gigas, Crassostrea angulata et Crassostrea virginica
Le marquage chromosomique en bandes G a été réalisé sur des chromosomes obtenus
à partir de tissu branchial de trois espèces d’huîtres creuses, Crassostrea gigas,
Crassostrea angulata et Crassostrea virginica, et a permis l’identification des dix
paires de chromosomes de ces espèces L’analyse comparée des caryotypes marqués
en bandes G a montré que les principales bandes G présentaient un modèle proche
chez les trois espèces, la paire de chromosome 3 étant identique Cependant quelques
différences ont pu être observées Les caryotypes de C gigas et C angulata ont révélé
plus de similitudes entre eux qu’avec celui de C virginica Ceci apporte un argument
supplémentaire à la divergence génétique entre ces deux lignées évolutives C gigas
et C angulata montrent un marquage en bandes G différent sur les deux bras de la paire du chromosome 7, ce qui corrobore leur séparation taxinomique L’application
de cette technique apporte une nouvelle approche pour la taxinomie et la génétique
des huîtres © Inra/Elsevier, Paris
chromosome / bandes G / Crassostrea gigas / Crassostrea angulata / Crassostrea
virginica
1 INTRODUCTION
Cytogenetic investigations in oysters were first mainly concerned with data
on chromosome number and gross morphology (e.g [1, 22]) Later,
morphome-tric analyses of karyotypes provided the characterisation of chromosome
mor-phology based on centromeric position (e.g [13, 17, 31, 37]) These studies
showed that oyster karyotypes were symmetrical and interspecific differences
consisted in the occurrence and differing proportions of metacentric and sub-metacentric chromosomes [18, 21].
The application of differential staining techniques, such as Ag-NORs for nucleolar organiser regions and C-banding for constitutive heterochromatin allowed the identification of some specific chromosome pairs in the karyotypes
of oyster species [14-16, 18, 19, 39] More recent techniques, such as fluorescent
in situ hybridization, have been tested in Crassostrea gigas [5, 9], and others, such as fluochrome staining and restriction endonuclease treatment, have been carried out in other bivalve species [25, 26] But although the data obtained
using these differential staining techniques provide a better knowledge of
the karyotypes of bivalve species, they do not allow the identification of all individual chromosomes
The G-banding technique, defined as a system of alternating dark and light bands throughout the length of the euchromatic parts of chromosomes [35],
allows the identification of each individual chromosome which enables one to
prepare precise and detailed karyotypes This technique has been routinely used
in vertebrate cytogenetics, especially in mammals (e.g [11, 12, 34, 41]) Only
a few studies have focused on lower vertebrates, such as fishes (e.g [2, 6, 7,
24]) and on invertebrates such as insects (e.g [4, 23, 33]) Among bivalves, G-banding patterns have been attempted in Mytilus [25, 27] and in the oyster
Crassostrea virginica [32].
In this study, G-banding patterns are described in three cupped oys-ters: Crassostrea gigas (Thunberg), the Pacific oyster, Crassostrea angulata
(Lamark), the Portuguese oyster and Crassostrea virginica (Gmelin), the
East-ern American oyster.
Trang 32 MATERIALS AND METHODS
2.1 Biological material
Specimens of each taxon were reared at the Ifremer hatchery in La Tremblade
(Charente-Maritime, France) Specimens of Crassostrea gigas were collected from the Seudre estuary, where this species was introduced from Japan [8] and
is currently farmed on a large scale Specimens of Crassostrea angulata were
collected in Setubal bay (Portugal), then acclimated at the Ifremer hatchery. Their taxonomic status was assessed using mitochondrial DNA markers as
described in Boudry et al (3! Specimens of Crassostrea virginica were imported
from a wild population located in Shippagan, New Brunswick (Canada) and acclimated at the Ifremer hatchery These oysters were maintained in common
quarantine facilities until reproduction, and their progenies were sampled for chromosome analysis.
2.2 Chromosome preparation
Whole juvenile animals were incubated for 7-9 h in a 0.005 % solution of colchicine in seawater Because cell cultures are not yet available for molluscs,
we used growing somatic tissues such as gills as a source of mitoses After
dissection, gills were treated for 30 min in 0.9 % sodium citrate The material
was fixed in a freshly prepared mixture of absolute alcohol and acetic acid (3:1)
with three changes of 20 min each Slide preparations were made from pieces
of gill tissue from each individual, using an air drying technique (38!.
2.3 G-banding
G-banding was performed by the ASG method (acetic/saline/Giemsa) after Sumner et al [36] Chromosome preparations were treated for 1 h at 60 °C
in 2 x SSC (0.3 M sodium chloride: 0.03 M trisodium citrate) After rinsing
in distilled water, the slides were stained with 2 % Giemsa-stain in phosphate
buffer, pH 6.8 for 90 min Best results were obtained by banding within
5-10 days of chromosome preparation.
Photographs of G-banded metaphases were taken with a Zeiss III photomi-croscope Karyotypes were made on the basis of length, centromeric position
and banding pattern Because we were working with somatic tissues, we had to
use many animals to obtain a sufficient number of mitoses Moreover, the
lat-ter showed different levels of condensation making the number of cells we could work on even smaller Thus, in total, 18 G-banded karyotypes were examined
in C gigas, 20 in C angulata and 21 in C virginica.
3 RESULTS
Establishing a repetitive G-banding pattern requires a similar degree of condensation of chromosomes to be compared Although a large number of
metaphases was observed, we selected only those with similar degrees of condensation for interpretation.
Trang 4Figure 1 presents example of G-banded metaphase of of the three species studied, C gigas In figure 2, haploid G-banded karyotypes are
shown to facilitate comparison of homologous chromosome pairs between the three species studied The karyotype of C gigas consists of ten metacentric
chromosomes, that of C angulata has nine metacentric and one submetacentric
(no 8) and the karyotype of C virginica includes eight metacentric and two
submetacentric (nos 4 and 8) chromosomes !18).
Figure 3 gives a schematic representation of the G-banding patterns obtained
for the three species Owing to differences in condensation of chromosomes only
Trang 5major G-bands used to compare the three species, with emphasis their number rather than their position.
Chromosome 1: on the short arm, both C gigas and C angulata show two
major bands, while in C virginica four major bands are present All three
species present four bands on the long arm.
Chromosome 2: on the short arm, C gigas shows three bands while in C
angulata and C virginica two major bands are present On the long arm, the three species are characterised by two major bands
Chromosome 3: the pattern is very similar across the three species, being
characterised by two major bands at the extremities of the short arm and one
large band at the centre of the long arm.
Chromosome 4: on the short arm, in C gigas and in C angulata, the bands
are observed in subtelomeric and near centromeric positions while C virginica
is characterised by one centrally located major band Four bands are located on
the long arm of the three species differing slightly in their position, which was
probably caused by the different degrees of condensation of the chromosomes
Chromosome 5: the three species present three bands on the short arm On the long arm, the G-banding pattern is different between the three species.
Trang 6Chromosome 6: on the short arm, three bands observed in
species On the long arm, C gigas and C angulata show four successive bands,
while in C virginica the banding pattern is characterised by the presence of
only one major median band
Chromosome 7: C gigas shows two bands on the short arm and two on the long arm In C virginica and C angulata, three bands are seen on both the short arm and the long arm These differ slightly in intensity and position.
Chromosome 8: the short arm in all three species is characterised by the
presence of one major band There are three major bands on the long arm of
C gigas and C angulata and four bands in C virginica.
Chromosome 9: on the short arm, two bands are seen in the three species.
On the long arm, C gigas presents three near equivalent bands differing from
C angulata and C virginica which are characterised by the presence of one
major band
Chromosome 10: the three species are characterised by two bands at the extremities of the short arm Two major bands are seen on the long arm of C
gigas and C angulata while three are present in C virginica.
4 DISCUSSION
The application of G-banding to three species of oysters: C gigas, C
angulata and C virginica, allowed individual identification of the chromosomes which makes it possible to prepare accurate karyotypes and describe the
respective idiograms.
Comparison with previous G-banding analysis of C virginica [32] showed that the number of G-bands identified as black bands (i.e the most distinct)
was similar in chromosome pairs 5 and 9 but quite different in the remaining
chromosome pairs This can be explained by i) different karyotypes of the
oys-ter populations studied (6 m-4 sm in Tabasco, Mexico; 8 m-2 sm in our
popula-tion), ii) different techniques used, iii) different condensation of chromosomes The major G-bands of the three Crassostrea species studied here show a
sim-ilar pattern on the whole chromosome 3, on the short arms of chromosomes 5,
6, 8, 9 and 10 and on the long arms of chromosomes 1, 2 and 4 C gigas and
C angulata present additional similarities on the short arms of chromosomes 1
and 4, and on the long arms of chromosomes 6, 8 and 10 These two taxa, often considered as the same species !28!, have been differentiated by mitochondrial DNA analysis [3, 30] and karyotype analysis [18] G-banding highlights
sim-ilarities between these two taxa, except for chromosome 7 where both arms are shown to be different This difference corroborates their taxonomic
sepa-ration C angulata and C virginica also display additional similarities in the number of major G-bands on the whole chromosome 7, the short arm of
chro-mosome 2 and the long arm of chromosome 9, but they differ on the short
arms of chromosomes 1 and 4, and on the long arms of chromosomes 5, 6, 8 and 10 Karyological differences between these two species have been previ-ously observed !18! C virginica contrasts with C gigas on the short arms of chromosomes 1, 2, 4 and 7, and on the long arms of chromosomes 5, 6, 7, 8, 9 and 10 Genetic divergence between C gigas and C virginica has been demon-strated by molecular phylogenies [20, 29] and by karyotype analysis !18! The
Trang 7differences G-banding pattern between C gigas and C virginica substantiate their genetic difference
Therefore, from the analysis of the banding karyotypes of the three species,
we can conclude that they generally resemble each other with chromosome pair
3 being similar in all the three species However, differences were observed from
one species to another, showing that there is a higher resemblance between the banded karyotypes of C gigas and C angulata than between these two species
and C virginica.
Because of the economic and ecological importance of oysters, genetic inves-tigations are of special interest In this respect, the identification of structural chromosomal features could be very useful in gene mapping, hybrid breeding
or stock conservation programmes The individual identification of the
chro-mosomes by G-banding will provide a better knowledge of the aneuploidy
phe-nomenon reported in oysters (e.g [40]) by identifying missing chromosomes
Similarly, G-banding could also provide a very valuable technique for
chromo-some segregation studies on triploid and tetraploid oysters [10] The applica-tions of G chromosome banding are therefore numerous and represent a useful
new tool in oyster genetics.
ACKNOWLEDGEMENT
This work was supported by a French-Portuguese co-operation (no 158 C 1),
by a research training project (contract no FAIR GT 97-3599) and part
of Genephys program (Contract no FAIR 95-421) We thank S Sabini and
S Heurtebise for excellent technical assistance and H McCombie for advice on
the English.
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